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Liver tissue engineering is promising as an alternative strategy to treat liver failure. However, generating functional hepatocytes from stem cells is conventionally restricted by the immature status of differentiated cells. Besides, embedding hepatocytes in bulk scaffold is limited by a lack of vascularity and low cell-packing density. Here, we fabricate collagen type I (COL1) microspheres for efficient hepatic differentiation of pluripotent stem cells and subsequent assembly of prevascularized liver tissue (PLT). Using a microfluidic platform, we demonstrate that hydrogel COL1 microspheres (mCOL1) encapsulating human embryonic stem cells (hESCs) can be reproducibly generated and efficiently differentiated into hepatocyte-like cells (HLCs) microspheres for the first time. Compared with other culture configurations such as encapsulation of hESC in a bulk COL1 hydrogel and 2D monolayer culture, mCOL1 with high uniformity produce HLC microspheres of improved maturity based on comprehensive analyses of cell morphology, transcriptome profile, hepatic marker expression and hepatic functions. In addition, these HLC microspheres can be applied as building blocks to self-assemble with endothelial cells to construct a dense PLT. The PLT resembles native liver tissue with high cell-packing density, shows successful engraftment in mice liver following implantation, and exhibits improved hepatic function in vivo. Overall, it is believed that this multiscale technology will advance the fabrication of stem cell-based liver tissue for regenerative medicine, drug screening, and in vitro liver modeling.

Articular cartilage plays an important role in human body. How to repair articular cartilage defects when they appear due to various factors has always been a major clinical challenge. Recently, studies have shown that slowing the degradation of cartilage extracellular matrix (ECM) and modulating the inflammatory response of the host thereby promoting cartilage tissue regeneration are important in the cartilage repair process. In this study, a drug-loaded injectable hydrogel was constructed for repairing articular cartilage. This hydrogel could not only maintain the phenotype of chondrocytes but also regulate the inflammatory response of the host. The injectable sodium alginate (SA)/bioglass (BG) hydrogel was mixed with the injectable thermal-responsive SA/agarose (AG)/quercetin (Que) hydrogel to obtain an injectable hydrogel containing both Que and BG (Que-BG hydrogel) for articular cartilage regeneration. The Que-BG hydrogel has a proper swelling ratio that can promote integration between the formed tissue and host tissue, and it allows Que to release slowly

The folding of tissues/organs into complex shapes is a common phenomenon that occurs in organisms such as animals and plants, and is both structurally and functionally important. Deciphering the process of tissue folding and applying this knowledge to engineer folded systems would significantly advance the field of tissue engineering. Although early studies focused on investigating the biochemical signaling events that occur during the folding process, the physical or mechanical aspects of the process have received increasing attention in recent years. In this review, we will summarize recent findings on the mechanical aspects of folding and introduce strategies by which folding can be controlled in vitro. Emphasis will be placed on the folding events triggered by mechanical effects at the cellular and tissue levels and on the different cell- and biomaterial-based approaches used to recapitulate folding. Finally, we will provide a perspective on the development of engineering tissue folding toward preclinical and clinical translation. Statement of significance Tissue folding is a common phenomenon in a variety of organisms including human, and has been shown to serve important structural and functional roles. Understanding how folding forms and applying the concept in tissue engineering would represent an advance of the research field. Recently, the physical or mechanical aspect of tissue folding has gained increasing attention. In this review, we will cover recent findings of the mechanical aspect of folding mechanisms, and introduce strategies to control the folding process in vitro. We will also provide a perspective on the future development of the field towards preclinical and clinical translation of various bio fabrication technologies.

Osteochondral regeneration with the formation of hyaline cartilage and subchondral bone as well as the integration between the newly formed tissues with the host tissue still remains a great challenge. In this study, a construct containing an injectable continuous stratified scaffold and multiple cell systems was designed for enhancing osteochondral regeneration. Briefly, an injectable sodium alginate(SA)/bioglass (BG) composite hydrogel containing bone marrow stem cells (BMSCs) (SA/BG + BMSCs) was used for subchondral bone regeneration and an injectable thermosensitive SA/agarose (AG) composite hydrogel with co-culture of BMSCs and articular chondrocytes (ACs) (SA/AG + ACs/BMSCs) was applied for articular cartilage regeneration. The continuous SA phase and the stratified structure enable the scaffold to mimic the natural osteochondral structure. In addition, the SA/BG + BMSCs hydrogel could enhance the osteoblast differentiation of BMSCs by upregulating their alkaline phosphatase and collagen I gene expressions, and the SA/AG + ACs/BMSCs hydrogel could promote the chondrocyte differentiation of BMSCs by upregulating their Acan and collagen II gene expressions, which indicated that this stratified scaffold could mimic the natural osteochondral function. Furthermore, after the stratified construct was injected into a rat osteochondral defect model, obvious neonatal articular cartilage tissues and subchondral bone tissues with regular surface and highly integration with normal tissues could be observed. This structural and functional biomimetic construct, together with its proper swelling ratio, could not only stimulate the hyaline cartilage and subchondral bone regeneration in an entire osteochondral unit but also promote the integration between the newly formed tissues and the host tissue.

Recent studies have unveiled the unique osteogenesis and angiogenesis abilities of akermanite. However, such bioceramics rarely fit well into irregularly shaped bony cavities (such as bone defects and the maxillary sinus). In this study, an injectable hydrogel SAG system containing sodium alginate, akermanite and glutamate was prepared and evaluated in such defects. Cell proliferation experiments showed that except for stoste, the other extracts showed no cytotoxicity at various concentrations. In addition, the gene expression of Runx2 (runt-related transcription factor-2), BMP-2 (bone morphogenetic protein-2), ALP (alkaline phosphatase), and BSP (bone sialoprotein) and the activity of ALP were significantly higher in human bone marrow stromal cells (hBMSCs) cultured with 1/2 hydrogel extracts containing 66.9 ppm calcium ions, 23.8 ppm magnesium ions and 33.5 ppm silicon ions compared to those in hBMSCs cultured with the control medium, which indicated that the hydrogel extracts could stimulate the osteogenic differentiation of hBMSCs. Further experiments revealed that the ERK signaling pathway was engaged in osteogenic differentiation as early as 15 minutes after incubation with the hydrogel extracts. In addition, hBMSCs incubated in half-diluted extracts exhibited an almost doubled migration ability compared with the hBMSCs in the control group. More specifically, compared to the control group, hBMSCs cultured with 1/2 hydrogel extracts showed a 7% increase and a 10-fold increase in the protein and gene levels of CXCR4 (C-X-C chemokine receptor type 4), respectively. In vivo tests demonstrated that the bone formation rate of SAG hydrogels injected alone was similar to that of its counterpart seeded with BMSCs, reaching 24% at three months after operation. Therefore, it could be concluded that SAG hydrogels contribute to bone regeneration by not only promoting osteogenic differentiation but also by enhancing the recruitment of BMSCs to defect sites, making the injectable SAG hydrogels competent for the regeneration of irregular bony cavities.

Three-dimensional (3D) culture techniques, such as spheroid and organoid cultures, have gained increasing interest in biomedical research. However, the understanding and control of extracellular matrix (ECM) effect in spheroid and organoid culture has been limited. Here, we report a biofabrication approach to efficiently form uniform-sized 3D hepatocyte spheroids and encapsulate them in a hybrid hydrogel composed of alginate and various ECM molecules. Cells were seeded in a microwell platform to form spheroid before being encapsulated directly in a hybrid hydrogel containing various ECM molecules, including collagen type I (COL1), collagen type IV (COL4), fibronectin (FN), and laminin (LM). A systematic analysis of the effect of ECM molecules on the primary mouse hepatocyte phenotype was then performed. Our results showed that hydrogel encapsulation of hepatocyte spheroid promoted hepatic marker expression and secretory functions. In addition, different ECM molecules elicited distinct effects on hepatic functions in 3D encapsulated hepatocyte spheroids, but not in 2D hepatocyte and 3D non-encapsulated spheroids. When encapsulated in hybrid hydrogel containing LM alone or COL1 alone, hepatocyte spheroids exhibited improved hepatic functions overall. Analysis of gene and protein expression showed an upregulation of integrinα1 and integrinα6 when LM was introduced in the hybrid hydrogel, suggesting a possible role of integrin signaling involved in the ECM effect. Finally, a combinatorial screening was performed to demonstrate the potential to screen a multitude of 3D microenvironments of varying ECM combinations that exhibited synergistic influence, indicating a strong positive effect of COL1 and a negative interaction effect of COL1·LM on both albumin and urea secretion. These findings illustrate the broad application potential of this biofabrication approach in identifying optimal ECM composition(s) for engineering 3D tissue, and elucidating defined ECM cues for tissue engineering and regenerative medicine.

Biomaterial topography-based strategies are regarded as an effective way to regulate the osteoimmune environment which plays an indispensable role in the bone regeneration process. The rapid development of manufacture techniques makes it possible to investigate the cell-topography interactions by preparing various micro and nano-topographical surfaces on biomaterials. Still, it is a challenge to prepare well-defined micro/nano hierarchical structures of bioceramics due to the inherent brittleness of ceramic materials. Also, the correlation between osteoimmunomodulation initiated by micro/nano hierarchical topographies and the tissue regeneration outcomes is unclear. In this study, we prepared well-defined micro/nano hierarchical structures on hydroxyapatite (HA) bioceramics through the combination of the photolithography and hydrothermal techniques. Three different microscale circular patterns (4 μm, 12 μm and 36 μm) and nanotopographies (nanoneedle, nanosheet and nanorod) were fabricated by changing the size of the mask and the condition of the hydrothermal reaction. The macrophage responses on the nanoneedle structures with different micropatterns were investigated and the micro/nano hierarchical structures with appropriate pattern sizes could either promote or alleviate the macrophage polarization, which further affected the outcomes of the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) and angiogenic activity of human umbilical vein endothelial cells (HUVECs). Our study demonstrated that osteoimmunomodulation could be manipulated via tuning the micro/nano hierarchical structures, which could lead to a new strategy for the development of bone biomaterials with favorable osteoimmunomodulatory properties.

Akermanite (Aker) has been widely used for bone regeneration through regulating osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). Previously, we developed an injectable Aker/sodium alginate (Aker/SA) hydrogel to facilitate bone regeneration. However, the effect of this injectable hydrogel on the in vivo response, particularly the inflammatory response, has not been fully understood. Here, to elucidate the response following the implantable of Aker/SA hydrogel, we investigated the interaction among Aker/SA hydrogel, inflammatory cells and cells involved in bone regeneration (BMSCs). Specifically, we cultured macrophages (RAW 264.7 cell line) with the extract liquid of Aker/SA and assessed their phenotypic changes. Subsequently, BMSCs (2 × 105 cells per 24 well) were cultured with different conditioned media including that of Aker/SA hydrogel-activated macrophages to investigate their effect on cell migration. Finally, Aker/SA hydrogel was injected subcutaneously (1 × 106 cells ml−1) in rat to verify its effect in vivo. The in vitro results indicated that Aker/SA hydrogel activated macrophages towards M2 phenotype and stimulated macrophages to express anti-inflammatory factors. In addition, the conditioned medium collected from Aker-activated macrophages could accelerate the migration of BMSCs in 24 h. Consistent with the in vitro results, when the Aker/SA hydrogel was injected subcutaneously, more M2 macrophages could be observed than when the SA solution was injected after 7 d. Besides, when BMSCs were delivered via subcutaneous injection, more BMSCs were recruited by the Aker/SA hydrogel than the SA solution. All these results suggest that the Aker/SA hydrogel can modulate the immune environment at the implantation site and subsequently recruit BMSCs, which can be one of the mechanisms through which the Aker/SA hydrogel accelerates new bone formation.

Heart failure (HF) is a common and serious manifestation after myocardial infarction (MI). Despite their clinical importance, current treatments for MI still have several limitations. Revascularization has been proven to have positive effects on MI-induced damage. Currently biomaterial-based angiogenesis strategies represent potential candidates for MI treatment. Bioglass (BG) is a commercially available family of bioactive glasses. BG has angiogenic properties and thus might be an attractive alternative for MI treatments. Here, we loaded BG in sodium alginate (BGSA), locally injected it into peri-infarct myocardial tissue and examined its suitability for inducing cardiac angiogenesis and eventually improving cardiac function following MI. Cardiac function was evaluated via echocardiography. Infarct morphometry, angiogenesis, apoptosis and angiogenic protein expression were all analysed 4 weeks after BGSA injection. Compared with the control treatment, BGSA was sufficient to prompt angiogenesis, suppress apoptosis, up-regulate the expression of angiogenic proteins, attenuate infarct size, preserve wall thickness and eventually improve cardiac function. Our results demonstrate the feasibility and effectiveness of BGSA in myocardial regeneration via angiogenesis, suggesting that BGSA is a potential therapeutic strategy for post-infarct myocardial regeneration.